GM Crop Database

Database Product Description

1345-4 (1345-4)
Host Organism
Solanum lycopersicum (Tomato)
Trait

Increased shelf-life (delayed ripening) due to reduced ethylene accumulation through introduction of truncated aminocyclopropane cyclase (ACC) synthase gene.

Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
DNA Plant Technology Corporation

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1995 View
Mexico 1998 1998
United States 1994 1994 1995

Introduction Expand

Tomato line 1345-4 was developed using recombinant DNA techniques to express the trait of delayed ripening of tomato fruit. The transgenic line contains a truncated version of the tomato 1-aminocyclopropane-1-carboxyllic acid (ACC) synthase gene, normally found in tomato. This endogenous enzyme is responsible for the conversion of s-adenosylmethionine to ACC, which is the immediate precursor of ethylene, a phytohormone known to play a key role in fruit ripening. The presence of the truncated ACC synthase gene suppresses the normal expression of the native ACC synthase gene, and while not completely understood, the mechanism of “downregulation” is likely linked to the coordinate suppression of transcription of both the endogenous gene and the introduced truncated ACC synthase gene. The in situ accumulation of ethylene in the transgenic tomatoes was only about 1/50 the level found in the unmodified parental line and the fruit does not fully ripen unless an external source of ethylene is applied. An antibiotic resistance marker gene (neo) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of this transgenic tomato. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the neo gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
ACC 1-aminocyclopropane-1-carboxylate synthase DR

CaMV 35S

A. tumefaciens nopaline synthase (nos) 3'-untranslated region

3

Truncated, sense orientation

nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens octopine synthase 3 Native

Characteristics of Solanum lycopersicum (Tomato) Expand

Center of Origin Reproduction Toxins Allergenicity

The regions of Ecuador, Peru, and the Galapagos Islands.

Almost exclusively self-pollinating; hybridization with related Solanum species (e.g., S. lycopersicoides) requires human intervention.

Glycoalkaloids, primarily alpha-tomatine, but also solanine and chaconine. Also, lectins and oxalate.

Although not a major cause of allergic reactions, several glycoproteins from tomatoes are known to be allergenic.

Modification Method Expand

The transgenic tomato line 1345-4 was produced by Agrobacterium-mediated transformation of the parental line 91103-114 (Lycopersicon esculentum var. esculentum) in which the transfer-DNA (T-DNA) region of the bacterial tumour inducing (Ti) plasmid was modified to contain DNA sequences encoding a truncated ACC synthase gene construct from the tomato cultivar ‘Golden Nugget’ and the NPTII encoding neo gene from E. coli strain K12. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome. Expression of the ACC synthase gene was under the regulatory control of the 35S promoter from the cauliflower mosaic virus (CaMV) and the 3’-polyadenylation signal from the nopaline synthase gene (nos) from A. tumefaciens. Expression of the neo gene was under the control of the nos promoter sequence and octopine synthase termination sequence from A. tumefaciens.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analyses of genomic DNA from line 1345-4 demonstrated the insertion of three copies of the T-DNA at a single insertion site. There was no incorporation of translatable plasmid DNA sequences outside of the T-DNA region.

Genetic Stability of the Introduced Trait

It was determined that tomato line 1345-4 was stable and that the introduced genes segregated as a single locus in a Mendelian fashion.

Expressed Material

The only novel protein expressed in tomato line 1345-4 was the enzyme NPTII, which was expressed at very low levels ranging from 0.72 to 2.36 µg/mg fresh weight of tomato. While transcription of the truncated ACC synthase gene was detected in transgenic tomato tissues, the partial gene did not express a protein product.

Environmental Safety Considerations Expand

Field Testing

The transgenic tomato line 1345-4 was field tested in the United States from 1992 to 1994. The agronomic characteristics of line 1345-4 were evaluated extensively in laboratory, greenhouse, and field experiments. The line 1345-4 retained the agronomic characteristics of the parental tomato, and field reports on seed germination rates, yield characteristics, disease and pest susceptibilities, and fruit compositional analyses determined that line 1345-4 was comparable to the unmodified parental line, with the exception of reduced ACC synthase activity. Field trial reports demonstrated that transgenic tomato line 1345-4 did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.

Outcrossing

Cultivated tomatoes are self-fertile, and almost exclusively self-pollinating. Their unique flower and anther morphology makes tomato an essentially cleistogamous plant (self-pollination and fertilization occur within an unopened flower). A low crossing rate between tomato varieties was demonstrated and attributed to the limited availability of pollen and poor foraging activity of insect pollinators. Several related species are found as weeds in tomato fields, however tomato (Lycopersicon esculentum var. esculentum) is generally sexually incompatible with all these weedy relatives. Two Solanum species, S. lycopersicoides and S. rickii, can be crossed with commercial tomato under specific, controlled conditions, but they do not naturally cross with L. esculentum and require the intervention of man. Neither Solanum species is a weed pest in the United States. The cherry tomato, L. esculentum var. cerasiforme, can be crossed with tomato. However, it would be very unlikely for traits in transgenic tomato line 1345-4 to naturally introgress into cherry tomatoes (var. cerasiforme) in the United States since the rate of outcrossing in tomatoes is low and cherry tomatoes are not common in areas devoted to large-scale cultivation of tomatoes. It was concluded that the chance of genetic exchange both among tomato crops and to other species was remote. In the event that an outcrossing event involving pollen from tomato line 1345-4 did occur, it was unlikely the modification to delay fruit softening would affect seed persistence or weediness potential in progeny.

Weediness Potential

Tomatoes are not considered a weed pest. There was no evidence to indicate that the presence of a truncated ACC synthase gene in line 1345-4 would convert it into a weed. Tomato volunteers are not uncommon, but are easily controlled using herbicides or by mechanical means. Seed dispersal by birds or mammals is insignificant. Furthermore, due to its tropical origin, tomato is very sensitive to temperatures below 10°C and winter cold will kill the majority of volunteer seedlings following harvest. Tomatoes are not persistent in undisturbed environments without human intervention. It was concluded that there was no likelihood that tomato line 1345-4 would have enhanced weediness traits compared to non-transformed tomatoes.

Secondary and Non-Target Adverse Effects

It was concluded that the genes inserted into tomato line 1345-4 would not result in any deleterious effects or significant impacts on nontarget organisms, including those that are recognized as beneficial to agriculture and those that are recognized as threatened or endangered in the United States. The truncated ACC synthase gene did not result in the expression of any new protein(s) in line 1345-4 and should not have any toxic properties.

Impact on Biodiversity

The transgenic tomato line 1345-4 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of tomato production. Since the risk of gene transfer to wild relatives in the United States is very remote, it was determined that the risk of transferring genetic traits of line 1345-4 to species in unmanaged environments was not significant.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The human consumption of the modified tomato line 1345-5 will be as both fresh and processed tomato products. Fresh market tomatoes are eaten whole and sliced or diced in a variety of foods. Processed tomatoes are consumed in the form of soups, preserves, ketchup, paste and prepared sauces. The genetic modification of these novel hybrids will not result in any change in the consumption pattern for fresh or processed tomato products. Line 1345-4 is expected to replace other tomato cultivars currently in use due to improved quality and handling characteristics. Hence, it will provide an alternate or additional choice to consumers and food manufacturers.

Nutritional Data

The analysis of nutrients from the novel 1345-4 line and the non-transgenic 91103-114 parental line did not reveal any significant differences in the levels of macro- and micronutrients. The presence of NPTII protein was judged to be insignificant with respect to any human health risk due to exposure. The consumption of this product will, therefore, have no significant impact on the nutritional quality of the food supply in Canada and the United States. Tomatoes produce naturally occurring glycoalkaloids, alpha-tomatine being the most common. The level of alpha-tomatine decreases as the fruit matures, so that the amounts in vine-ripened red tomatoes are negligible. The concentration of alpha-tomatine in immature tomatoes from transgenic line 1345-4 was 1.13-1.37 mg/100g fresh weight tissue, as compared with 3.37-3.59 mg/100g fresh weight tissue for the unmodified 91103- 114 parental line. However, these levels evened out once the fruit ripened into mature red tomatoes. In vine-ripened red tomatoes, the amount of alpha-tomatine in transgenic tomatoes was 0.11/100g fresh weight tissue compared to 0.07 mg/100g fresh weight tissue in the non-transgenic parental line. Other than reduced ACC synthase activity, the disease, pest and other agronomic characteristics of the transgenic 1345-4 line were comparable to the unmodified parental line.

Toxicity and Allergenicity

The reduced synthesis of native ACC synthase was judged not to have any potential for additional human toxicity or allergenicity. The truncated ACC synthase gene was not expressed in line 1345-4 and should not have any toxic or allergenic properties. The NPTII protein is widespread in nature and no toxic or allergenic effects have ever been reported.

Abstract Collapse

The tomato is a vine-like herb of the nightshade family (Solanaceae) that also includes potatoes, peppers and eggplants. Botanically this vegetable is a fruit (a berry), which although being a perennial plant in the tropics, is grown as an annual plant in northern climates. The tomato is a native of the Peru, Bolivia, and Ecuador area of the Andes Mountains. Its antiquity is uncertain in regard to cultivation but it was being cultivated when Europeans discovered America. However, it was not generally cultivated in the United States until 1835 because, until then, it was widely believed to be poisonous. Tomatoes are propagated from seeds. In temperate regions seeds are generally started in greenhouses, hotbeds, or cold frames; the plants are set out in the fields when danger of frost is past. Fresh tomatoes are harvested by hand, while those destined for canning or for processing into soups, sauces and ketchups are harvested by machine. The numerous varieties differ greatly in plant form and fruit type, the latter ranging from a small currant size through cherry, plum, and pear forms to the large, nearly round fruits, 10 cm (4 in) or more in diameter, which are the most widely grown. All forms include red- and yellow-fruited varieties. Tomatoes are a valuable source of food minerals and vitamins, and are low in calories. One medium-sized tomato provides 57% of the recommended daily allotment (RDA) of vitamin C, 25% RDA vitamin A, and 8% RDA iron, yet it has only 35 calories. Tomatoes are also rich in an anti-oxidant called lycopene, a carotenoid that has been found to protect cells from oxidants that have been linked to cancer. In laboratory tests, lycopene was found to be twice as powerful as beta-carotene in neutralizing free radicals. Lycopene has been linked to risk reduction for a number of types of cancers, including prostate, lung and stomach, pancreatic, cervical, colorectal, oral and esophageal cancers. In the fresh market industry the tomato fruit is often picked at the mature green or breaker stages for long-distance shipping, and is then subsequently ripened by treatment with the gas, ethylene (12 to 18 h at 20ºC). For processing tomatoes the ethylene-producing compound, ethephon or Ethrel, is applied prior to harvest when only 10% of the fruit is ripe; this accelerates and concentrates fruit ripening and facilitates once-over machine harvest. Tomato line 1345-4 was developed using recombinant DNA techniques to express the trait of delayed ripening of tomato fruit. The transgenic line contains a truncated version of the tomato 1-aminocyclopropane-1-carboxyllic acid (ACC) synthase gene, normally found in tomato. This endogenous enzyme is responsible for the conversion of s-adenosylmethionine to ACC, which is the immediate precursor of ethylene, a phytohormone known to play a key role in fruit ripening. The presence of the truncated ACC synthase gene suppresses the normal expression of the native ACC synthase gene, and while not completely understood, the mechanism of “downregulation” is likely linked to the coordinate suppression of transcription of both the endogenous gene and the introduced truncated ACC synthase gene. The in situ accumulation of ethylene in the transgenic tomatoes was only about 1/50 the level found in the unmodified parental line and the fruit does not fully ripen unless an external source of ethylene is applied. Tomato line 1345-4 was field tested in the United States from 1992 to 1994. The agronomic characteristics of line 1345-4 were evaluated extensively in laboratory, greenhouse, and field experiments. Line 1345-4 retained the agronomic characteristics of the parental tomato, and field reports on seed germination rates, yield characteristics, disease and pest susceptibilities, and fruit compositional analyses determined that this line was comparable to the unmodified parental line, with the exception of reduced ACC synthase activity. Field trial reports demonstrated that transgenic tomato line 1345-4 did not exhibit weedy characteristics, and had no effect on non-target organisms or the general environment. The transformed tomato line was not expected to impact on threatened or endangered species. Cultivated tomatoes are self-fertile, and almost exclusively self-pollinating. Their unique flower and anther morphology makes tomato an essentially cleistogamous plant, in which self-pollination and fertilization occur within an unopened flower. A low crossing rate between tomato varieties was demonstrated and attributed to the limited availability of pollen and poor foraging activity of insect pollinators. Several related species are found as weeds in tomato fields, however, commercial tomato is generally sexually incompatible with these weedy relatives. Two Solanum species, S. lycopersicoides and S. rickii, neither of which is a weed pest in the United States, can be crossed with commercial tomato only under specific, controlled conditions requiring human intervention. The cherry tomato, L. esculentum var. cerasiforme can be crossed with tomato, L. esculentum var. esculentum. However, it would be very unlikely for tomato line 1345-4 to hybridize with cherry tomatoes in the United States since the rate of outcrossing in tomatoes is low and cherry tomatoes are not common in areas devoted to the large-scale cultivation of tomatoes. It was concluded that the chance of genetic exchange among tomato crops was small and outcrossing to other species, even more remote. In the event that an outcrossing event involving pollen from the transgenic tomato line 1345-4 did occur, it was unlikely that the delayed-ripening trait would increase the plant’s weediness or probability of survival. Tomatoes are consumed in both fresh (whole and sliced or diced in a variety of foods) and processed (soups, ketchup, paste, prepared sauces) forms, and the genetic modification introduced into the transgenic tomato line was not expected to result in any changes in consumption patterns. The analysis of nutrients from the novel 1345-4 line and the non-transgenic parental line did not reveal any significant differences in the levels of macro- and micronutrients. Levels of glycoalkaloids present in mature tomatoes from the transgenic line 1345-4 were within the range reported for conventional tomato varieties. The reduced synthesis of native ACC synthase was judged not to have any potential for additional human toxicity or allergenicity. The truncated ACC synthase gene was not expressed in line 1345-4 and should not have any toxic or allergenic properties.

Links to Further Information Expand

Office of Food Biotechnology, Health Canada U.S.Department of Agriculture, Animal and Plant Health Inspection Service US Food and Drug Administration USDA-APHIS Environmental Assessment

This record was last modified on Tuesday, September 15, 2015